Studies on the ultraviolet fluorescence of vitamin D and related compounds in acid-alcohol solutions

ANALYTIC.4L

BIOCHEMISTRY

Studies and

15,

on the Related

ANTHONY

%i-2%

(1966)

Ultraviolet Compounds

Fluorescence in Acid-Alcohol

J. PASSANNA4NTE

F,.orn the Division of Endocritlology. Near Jrrgcy College of Me~licir~e, Received

Decemhrr

.4ND

LOUIS

of

Vitamin

D

Solutions1 V. .4VIOL12

Depurtment oj Medicine, Jersey Cit !I? SPM: Jclscy 21, 1965

Despite the availability of a semiquantitative biological assay for the det,ermination of vitamin D-like activity in bioIogica1 fluids, the metabolic fate of vitamins Dz and D, in man is still unknown. The paucity of information is due primarily t,o lack of precise quantitative methodology for the identification of vitamin D and its metabolites. The technical limitations which prevent the physicochemical quantification of vitamin D in biological samples have been apparent for a quarter of a century (1, 2). Not only is t,he estimated concentration of vitamin D in human plasma prohibit.iveiy low for most chemical measurements (l), but, the problem is con~l~onnclccl by t,he relative abundance of structurally similar circulating steroids in plasma. Accordingly, the available chemical methods for vitamin D :maly,Gs have been limited t,o highly purified pharmaceutical preparations (3, 4). Our prevent interest in ihc biochcmi.qtry and metabolism of vitamin D led us to investigate the flilorescencc oi vitamin D ant1 related compounds. Sinre fluorescence annlysi-; has provctl to bc more ,senGtivc than iiltr:tviolet absorption methock in the analysis of biological matcrinls (5), our initial stutlies were itlainly conrcrnctl with the relative fluorescence in acid-alcohol solut,ions of ,
Jppamfus: 4-8106) with

The -4lnillco-~o~~l~~nn a 1P21 photomultiplicr

.qEct~ropllotofllloloruetcl

(Radio

Corporation

itllo~lcl

of ~4moric~:r)

288

l’ASS.4NN.4NTE

AS-D

AVIOLI

USCd for a11 measurements of activation and fluoreecencc ;;I)cctr:i and fluorometric tleternlin:ttions. ~tnndard ad Test &‘olcrtiom: Crystalline vitamin II, 3 or vitamin I ),, :I W’C~Cweighed into a volumetric flask and diluted with known amounts of absolute ethanol. Aliquots were then removed and diluted with 2Oc;/, sulfuric :tcitl-ethanol (\-,‘v I to establish the desired soIut,e concentr:ition in :I lO(//,, sulfuric acid-ethanol iv/v) mixture. The solutions were 1~1’e]):ll*ctl in htO~)~wlwl I-oluniet~ric flasks and placed in ;I water bath at, 75°C for I hr. I:pon withdr:~rval from the water hnth the flasks were air roolcd for I O-I 5 min. Tlrr solutions were subsequently transferred to f~~scd quartz cuvets i 1.2 cm square X 4.8 rin high) nntl their ultr:tviolct Auorcscence tlctc~rinincd JvitIi nctivntion at, 425 mp. Idcntic:~l prtpr;~tory ant1 fluorcswncc analytical xhemata were followed for crystalline l)ix’p:~rations of chol~~stcrol, 7-clcll;r-tlrocliolc~t~~rol, ergostcrol, tlili~drotncl~~~t~~t~ol, hydrocortisonc (cortkol) , c~or~tisonc. corticost,eronc, testostcronc, ~S~IUIK~, 17-/I-cstratliol, rstriol, n-a-tocophcrol, Ctnmin I<:: I ~Irnntlione) ( \-it,aniin A alcohol, t’rnns-p-carotene, fi,y-dipalmitoyl-L-~-lecithin, and tril)alnlitin.” Hw~zcrr~ Swum: 1.0 ml of fresh human serum was diluted ant1 n-cl1 ilkiecl with 4.0 ml of absolute alcohol in a conical centrifuge tube. The rrsuking nlisturc of I)rwipitnted protein and sulwrnat:tnt liquid was centrifuged :tt 2000 rI)nl for 10 nlin. The clc:~r sulwrnatant fluitl was dccnntcd into :I 10.0 nil Tolumetric flask and 5.0 ml of a freshly piq~:~recl 2Ocjr solution (1. ‘rj of c~onrentrntcd sulfuric acid-:ibsolute ethanol ntltlctl. The resulting .solution wns :Igit8ated to CIWIII‘C coml~lcto mixing ant1 clillltcd to ~olunw nit11 :~hbolutc c~thnnol. Sulwrjucnt, Iw:lting at. 7.5”(’ for 1 lir :md fluor~s~~‘nw tlctc,l.lrlin:ltion w-erc follonetl as outlined :thort*. \Y3R

t8,1. The Iritnltlin I)-cont:tining fractions wre pooled nnd the eluent solvents rcmovccl nit,h n Aream of nitrogen. The residue was then rcdissolved in :tb.solutc ctl~nol rind ~)rel~~~l for fluorescent analysis 2s dc.wrihetl above. RESULTS

tc:~citcrtio~r-~l~iot~(~.~(~(,~i~~Spectra: Figure 1 demonstrates the excitation and fluorwwncc qwctr:i of vitxmin I>,,. Alniost~ identical excitation and fluowsc~~n~~~ q)o(*t~x W(‘I’P obt:~inc~(l lvitll I-itamin I>?. For both T), and D.:,

WAVE

LENGTH

(“I&

E’rc. 1. 4l,tivation-fluolc,sI,ence spw~ra of vitamin D3 (n~~prosimstely acid-ethanol solIdion. The closed circles rind brolicn line reprosent trum, and the open circles rind solid line, fluorescence spectrum.

10 &ml) irl activation spcc-

Fluorescence-Concebation C~wves: .A plot of yit:rmin Dn concentration versu:: fluorc~ccncc is repre~wntccl in Fig. 2. A linear rwponee of fluoresccncc~ nit11 incrcnsing stcroitl conccntr:ltion wa,s fouiid in a range of O-50 ;rg,, ml. M‘hilc vitamin;-: I), and D:! have identical excitation and fluorescence ,qwt,ra I Fig. I), tlic specific fluorescence (i.e., unit2 ~~LIOI.C’Rcenrc/j~g/iul X 100) of D, at 475 111:~is approximately 20% higher than D, (Table 1). Blank readings, determined for the sulfuric acid-ethanol solvent system. rnngctl frolu 0.6-0.7 11.g‘ml in “vit:unin D,+ equiralcnts.” These blank xdues bare zubt~ruct~cd during all fluorescence determin:ttions. Fluorescence Spectra of Related Compounds: The existence of compounds structurally similar to ergocnlciferol and cholecalciferol in blood presents the problem of possible interfercncc due to the fluorescence generated by these mnterials. A variety of such compounds were, therefore, incubated with ,:ulfuric acid-ethanol at 75°C at concentrations

290

PASSAICNAKTE

FIG. 2. Fluorescence-concentration solutions. Activation at 425 mp and sent duplicate determinations.

FLUORESCENCE

MAXIMUM%

ACID-ETHANOL

AND

curve of vitamin D, in sulfuric fluorescence at 475 rnp. Concentric

TABLE 1 D AND

OF VITAMIN SOLUTIONS

AVIOLI

FOLLOWING

OTHER

SUBST.~NCES

ACTIVATION FlUOF2SCNLC~

Concentration. r/ml

Compound

Vitamin D, Vitamin D3 Vitamin A alcohol Tranx-fl-carotene Cholesterol 7-Dehydrocholesterol Ergosterol 7-p-Estradiol Estriol Dihydrotachysterol Cortisol Cortisone Corticosterone Testosterone Estrone P,r-Dipalmitoyl-n-c-lecithin Tripalmitin a Units

fluorescence/&ml

10 10 10 10 30,000 25 1.1 9r 25 9r 1.1 25 25 23 23 25 ‘25 200 100

acid-ethanol circles repre-

IN

AT 425

SULFURIC

mh

Specific

fluorescence

maximum, mP

itt 47.5 Inp, unit+

‘475 475

4.4 5.4 8.5 5.0 -5.0 x 10-a 2.0 2.4 0.06 01 0.0 0 0 0.0 0.0 0.0 0.0 0.0 0.0

475

Ai.5 530 520 310 530 530 NOM!

None KOlll2 None Kone None None ?Jone

X 100.

approximating those in human serum or higher. The fluorescence maxima and specific fluorescences at 475 rnp with activation at 425 rnp are depicted in Table 1. Of all the substances tested, only vitamin A alcohol and p-carotene

had identical fluorescence maxima (Table 1) and fluorescence spectra similar to Dz and DC: when nctivatcd at 425 ml* (Fig. 3). As noted in Tablt 1, the slwific flllorcscence~ of vitamin A and p-carotene at 475 n+ were approximately 1.6 ancl 0.9 tiulcs tllat of \-itamin D:,, respectively.

1

500

450 WAVE

FIG. 3. Fluorescence speclra ml), and vitamin D1 (1.6 pg/ml) rtt 425 mp. Vitamin d alcohol, the rloscd, open. and c*onventric

LENGTH

550

600

(m,u)

of vitamin A nlcohol (1 pg/ml), /3-carotene (1.3 pp/ in sulfuric acid-cthnnol solution following activation tr:zus-p-carotene, and Ctamin D, are represented 1)~ circles, rc~~wt~tirrly.

No iuenrurnble fluorcwenre \vad obtained for cholesterol solutions equivalent, to a normal wrum concentration of 3 mg/ml. At much higher but the concentrations 130 mg;,/ml) fluorcwence was noted at 475 1~11.~ fluorescence spectrum differed from vitamin D in that peak fluorescence occurred at 530 III/L. At concentrations of 25 pg,/ml both provitamins, 7-dehgdrocholesterol and ergostcrol: clisplaycd fluorescence at 475 111~ when activated at, 425 m/~, but, like cholrsterol, maximal fluorescence occurred at a higlwr wavelength, viz.. 520 m/~. The fluorescence at 520 n+ was 2.5 times grcatcr than that okrrctl at 475 nl!~. Despite different fluorescence masinla, t,lle specific fluorescences obtained with the provitamins at 475 I~;L were nppreciablc, 7-dehydrocholesterol and ergosterol fluorescence nvcrnging 37 :md 457: that, of vitamin D:;, rcspectirely (Table 1j . iUinirn,zl fluorcsccnce W:LSobtained at, 475 1~)” with 25 &ml solutions of 17-/3-estradiol and estriol and no fluorescence observed for similar concentrnt,ions of dih~d~otncl~yste~ol, cortisol, cortisone, corticosterone, testosterone, and cstronc. /3,r-clipalmitoyl-L-~~-lecit.hin and tripnlmitin, nonsteroid lipid constituents normally found in human plasma, were found not to fluoresce at concentrations of 200 and 100 /~g/ml, respectively. Fluoresceure of XorrstrporrificcDIe Collsfitrreuts of Hunmn Serum: Tile

tration equivalents ranged from 5.1 /~.g/ml of serum to 16.0, with a mean of 8.5, levels several hundred times those generally accepted for vitamin D and about t.en times the circulating plasma level of vitamin A (9). DISCUSSION

D,

The observed ultraviolet in lO$% (v/v) sulfrlric

fluorescence acid-ethanol

developed by vitamins I), and solutions supplements I’revious

observat’ions of Cohen ef trl. wherein citarnin D fluorescence in various acids and organic solxxbnts wm noted (2). ThiR ncid-nlcohol induced fluorescence sppe:trs to offer prolni~c for qumtitnting vit,aniin 11 in biological fluids if qwcific intcrfcrcnccs can be o~wzo~nc. The characteristic flllol.OsCCrlC(~ tlcrclolJc~(l :lt 475 III/J. following incuhtion at, 75°C and act~iwtion at 425 nip. has ~J(YW i~c~~~rocl~ic~~l ill IJotli ~JIII~~ solution an11 lluman r(w. The similarity of tllcb fl~~orwc~~nc~~ spwtra o~kaine(l for hot11 the vitamin I>-vit:min A ~IXJU~J il),. I) ,( ;\. ,8-c~nrotcncj is .striking. The st~ructurcs of t11cw cwln~Jon1lcll4 :~i’(l similar in tliat tllcy contain an open chin of ~On~llg:lt~Yd CkJll)Jk ~JOiids, thl‘w fOl’ ~~it~llllill 11, five for ~it.Xllin -4, and elcvcii for /I-c~:~rc~tcm~. :IS ~~liowi in 1’0ixi~11:1s 1. 11. :~ntl TTI. wspectivoly.

VITAMIN

D3

(I)

B-CAROTENE

VITAMIN

cm)

A ALCOHOL

(II,

294

PASSANNANTE

AND

AVIOL

ethanol extraction. The difference may be a reflection of the protein binding of vitamin D or related compounds described by Thomas et al. (10) a’ncl/or an enhanced extraction of p-carotene and vitamin D by the nonpolar chloroform solvent since lipid-soluble material cannot, be quantitatively extracted wit,h absolute ethanol. In this regard Kodicek has also noted that complete recovery of vitamin 1) from serum requires refluxing with ethanol i I I ) . The homogenization. saponification, and extraction Jnoceclurcs us:cd to prcJn~rc t,he srrmn s:un~~lcs for chromatography may have been more effcct,irc in removing protein-bound vit,amin D than thcx relat,ively mild alcoholics lwwiJ>itation of serum proteins. The present valuw obtained for iivitaitiin 1)” levels in hmuan plasma are obviously disJwoJ)ortion:ltt~ly higli and ~crvc only to illwtrat,e the numerous tcclrnical J)roblcme relating to thr extraction, isolation, and qunntitation of vitamin D in biological fluids. DcsJ)ite the reiuoval of provitamins 7-dehydrocholestcrol and ergosterol, which arc obvious interfering substances in the proposed acid-alcohol fluorescence system (Table I), t’hc valwx obtained are sevc~al hundred fl’jnes circulating vitamin I) lrvels as estimated by bioassay techniques (1, IO). The ohscrvcd fluoreswncc obtained wit.11 human wrum can only hc cxJ,lained to the extent of 2.5-30% based on the cstimatcrl fluorcscrncr of Jjhpsiological amounts of vitamin D, vitamin A, and p-carotcnc. It is possible that. ot,lier untreted structurally similar coinJ~ounds contribute to t,he ohscrvcd serum fluorcsccnrr at. 475 m/~ when activated at 425 m/b. The as yet, Iiniclentificd compounds wliich appear to Jnoducc at lewht sonic 70% of tlic “vitnniin D tyJw” Aiiorcwcnce may also rcprewnt rntirely mirelatctl biibstnncw or inctaholitc~ of vitninin D of tlict open-rhnin coiijIlgntcr1 double bond varkty (12-14). Thaw J~rrliminnry olwrvation s suggest: thCwfore, tlrat any quantitaf#ivc proccdurr basc~l on the flllOrWY~JlW of vitamin D in acid-alcohol not only of vitamin A and solutions must provirlc for t,lic scp:lration /3-cnrottnc ( 15) but, also of an unitlentific>d s1iJwtance (or suJwt:lnrcs) whic*li may signifirantly cont.rihutc to the olwrvcd fluorrscenw.

The nct,ivntioii :m(l flliorcscencc spectra of vit:Lniins D, rind I):: h:lr-e bwn csamincd nfttr eliciting fluorwwnw by heating in sulfuric acidrt,linnol solution. Curvilinear fluoi,cPcrncc-roncerlti.:~tion plots were oht.:tinerl for Dl ant1 D:: by activation at 42-5 m/r ~(1 mra~uremcnt of fluorcsccnw at 475 iii/~., i.e.. their. activation and fluowwcnw inasimn. rcspectiwly. Tllc fluorcwcnw of :I vnric+y of st~ructurally rchrtcd con~J~ound~ was nJ.w inr-wtip:~tcrl following artilxtion nt 42.5 nl/~. Of these, vitamin A and p-rarotcnc n‘c~w the only roinJ~oun& with silrlilxr fllwnwrcncc spwtrn.

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Rimsm,

K., And.

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